Abstract: Protein post-translational modifications play a key role in cellular signaling and metabolic regulation. Understanding this fundamental aspect of biology and its implications in disease pathology has emerged as a central goal of proteomics research. In pursuit of this goal, mass spectrometry is the analytical method of choice for large-scale identification of proteins and post-translational modification mapping and discovery. However, vast regions of protein sequence remain unmapped because they lack proteolytic cleavage sites. The proposed research program will address this critical shortcoming by in vitro evolution of orthogonal proteases that will introduce new cleavage sites and thereby sequence coverage directly in the vicinity of modified residues. A microfluidic compartmentalized in vitro selection strategy will be developed to survey 1010 mutant proteases simultaneously and enrich in highly active mutants that exhibit novel cleavage specificity. Enriched mutant pools will then be subject to more traditional high-throughput screening to identify mutants with extraordinary activity, robustness, and specificity. Employing this strategy, new proteases will be bred to cleave specifically at one of ten common post-translational modifications. These modifications are not only important in signaling and transcriptional regulation, but their chemical distinctiveness will maximize the probability of discovering mutant proteases capable of recognizing and cleaving adjacent these moieties. The mutant proteases will immediately generate complete sequence coverage for samples in the Scripps Florida pipeline, including viral regulatory proteins and cancer-related kinases that both harbor regulatory modifications in sequence coverage gaps. More broadly, the ability to conduct modification-specific protein digestions will transform mass spectrometry-based proteomics from a shotgun panning technology into a targeted search and discovery platform as each new protease tool is evolved. Public Health Relevance: Protein post-translational modifications are the chemical switches and knobs of metabolism, and their state and type teach us the molecular basis of cellular function and regulation. We are using evolution to generate new molecular tools that will specifically reveal the presence of these modifications, transforming mapping technology from a random panning expedition into a highly targeted survey. These targeted mapping campaigns will lead to more global understanding of how modifications control basic cellular processes and how diseases such as viruses and cancers hijack these controls.